Phylogenetic Analysis and Intrageneric Structure of the Genus Hyphornicrobium and the Related Genus Filornicrobium

International Journal of Systematic Bacteriology (1 998), 48,635-639 Printed in Great Britain

Phylogenetic analysis and intrageneric structure of the genus Hyphornicrobium and the related genus Filornicrobium

Frederick A. Rainey,' Naomi Ward-Rainey,' Christian G. Gliesche2 and Erko Stackebrandtl

Author for correspondence: Erko Stackebrandt. Tel: +49 531 26 16 352. Fax: +49 532 26 16 418. e-mail : erkoagbf-de

Deutsche Sammlung von Almost complete 16s rDNA sequences from the type strains of seven species of Mikroorganismen und the genus Hyphomicrobium and of Filomicrobium fusiforme have been Zellkulturen GmbH, D- 381 24 Braunschweig, determined. The Hyphomicrobium species form two phylogenetic clusters that Germany are only moderately related to each other. While cluster I contains the type Inst it ut fur Al lgemei ne species Hyphomicrobium vulgare, Hyphomicrobium aestuarii, Hyphomicrobium Mikro biologie, U n iversitat hollandicum and Hyphomicrobium zavarzinii, cluster II comprises Kiel, Am Botanischen Hyphomicrobium facilis, Hyphomicrobium denitrificans and Hyphomicmbium Garten 1-9, 0-241 18 Kiel, Germany methylovomm. Within the two species clusters, the species are highly related. Phylogenetically,Filomicmbium fusiforme clusters moderately with Hyphomicrobium species. The lack of distinguishing phenotypical properties presently excludes the possibility of describing cluster II as a new genus.

Keywords: Hyphomicrobium, Filomicrobium, intrageneric structure

INTRODUCTION been isolated and included in these and in taxonomic studies (Gebers et al., 1986; Gliesche et al., 1988; Hyphomicrobia are appendaged bacteria that repro- Stackebrandt et al., 1988; Roggentin & Hirsch, 1989; duce by budding and have a dimorphic life cycle Holm et al., 1996). involving non-motile prosthecate mother cells and motile swarmer cells (Hirsch, 1989). In contrast to The genus Hyphomicrobium presently contains nine morphologically similar taxa such as Hyphomonas species (Hirsch, 1989; Urakami et al., 1995), one of (Moore & Weiner, 1989), Pedomicrobium (Gebers, which, H. coagulans (Takada, 1975), is not available 1989), Dichotomicrobium (Hirsch & Hoffmann, 1989), from any culture collection, while H. indicum has been Filomicrobium (Schlesner, 1987) or Rhodomicrobium discussed in the literature to be a non-authentic (Imhoff & Truper, 1989), hyphomicrobia are restricted member of the genus (Hirsch, 1989; Urakami et al., facultative methylotrophs capable of growth on 1995). Of the other species, only a few strains have reduced C, compounds such as methanol, methylated been included in chemotaxonomic studies (Guckert et amines or formate (Harder & Attwood, 1978). In al., 1991 ;Sittig & Hirsch, 1992) and in the phylogenetic recent years, hyphomicrobia became of special interest analysis of 16s rRNA (Stackebrandt et al., 1988; because of their versatility and ability to use toxic Roggentin & Hirsch, 1989; Tsuji et al., 1990) and 5s waste compounds that are not metabolized by other RNA sequences (Stackebrandt et al., 1988; Boulygina methylotrophs (Hanson, 1992). They can be used in et al., 1993). These studies have indicated that the the denitrification of sewage (Nyberg et al., 1992) or genus belongs to the alpha subclass of the class drinking water (Liessnes, 1993) or in the bio- Proteobacteria. However, according to the results of remediation of C, compounds such as halomethanes, DNA dot-blot hybridization studies on hundreds of methyl sulphates and methylated phosphates (Large & Hyphomicrobium strains (Holm et al., 1996), 19 DNA Bamforth, 1988) and, consequently, many strains have similarity clusters were identified, which points towards the presence of a significantly higher number of species than presently described. This paper is dedicated to Dr Peter Hirsch on the occasion of his 70th birthday. In this study, we present a phylogenetic analysis on all The EMBL accession numbers for the sequences reported in this paper are available type strains of the genus Hyphornicrobium, Y14302-Y14313. which will allow subsequent affiliation of environ-

00698 0 1998 IUMS 635 F. A. Rainey and others

Table I. Bacterial strains analysed in this study

~~ Strain* Other designation(s)* Referencelsource

H. aestuarii IFAM NQ-521grT ATCC 27488 Hirsch (1989) H. denitriJicansDSM 1869T TK 0415 = IFAM HA-905 Urakami et al. (1995) H. facilis subsp. facilis IFAM H-526T DSM 1565, ATCC 27485 Hirsch (1989) H. facilis IFAM B-522 Hirsch (1 989) H. facilis subsp. tolerans IFAM I-551T ATCC 27489 Hirsch (1 989) H.facilis subsp. ureaphilum IFAM CO-582T ATCC 27492 Hirsch (1989) H. hollandicum IFAM KB-677T ATCC 27498 Hirsch (1989) H. methylovorum DSM 5458T ATCC 35216, KM-146 Hirsch (1989) H. vulgare IFAM MC-750T ATCC 27500 Hirsch (1989) H. zavarzinii IFAM ZV-622T ATCC 27496 Hirsch (1989) H. zavarzinii IFAM ZV-580 Hirsch (1989) F. fusiforme DSM 5304T Schlesner (1987) * ATCC, American Type Culture Collection, Rockville, MD, USA; DSM, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany ; IFAM, Institut fur Allgemeine Mikrobiologie, University of Kiel, Germany.

mental isolates to known species and the recognition additional strains of Hyphomicrobium were sequenced. of novel genomic nuclei that may represent novel Their EMBL accession numbers are : Hyphomicrobium species. vulgare IFAM MC-750T (Y 14302), Hyphomicrobium hollandicum IFAM KB-677T (Y 14303), Hypho- METHODS microbium aestuarii IFAM NQ-52 lgrT (Y 14304), Hyphomicrobium zavarzinii IFAM ZV-622T (Y 14309, Bacterial strains. Strains analysed in this study are listed in Hyphomicrobium zavarzinii IFAM ZV-580 (Y 14306), Table 1. Growth media and culture conditions followed Hyphomicrobium methylovorum DSM 545gT(Y 14307), described procedures for the cultivation of Hyphomicrobium Hyphomicrobium denitrijicans DSM 1869T (Y 14308), strains (DSM catalogue of strains, 1993, 1996). Hyphomicrobium facilis subsp. facilis IFAM H-526T 165 rDNA sequence determination and analysis. Extraction (Y 14 309), Hyphom icr obium facil is subsp . ureaph ilum of genomic DNA, PCR-mediated amplification of the 16s IFAM CO-582T (Y 143lo), Hyphomicrobium facilis rDNA and sequence analysis of the purified PCR products subsp. tolerans IFAM I-551T (Y1431 l), Hypho- were performed as described previously (Rainey et al., 1996), microbium facilis IFAM B-522 (Y14312) and Filo- and the sequence reactions were electrophoresed using a microbium fusiforme DSM 5304T (Y 14313). model 373A automatic DNA sequencer (Applied Bio- systems). The length of sequences ranged between 1410 and 1446 To determine the closest relatives of strains of hypho- bases, which corresponded to 91 and 94% of the microbia, their phylogenetic position was determined Escherichia coli sequence (Brosius et al., 1978), re- initially using the database ARB (Strunk & Ludwig, 1995). A spectively. The position of the type strain of the type fine resolution of the relatedness between hyphomicrobia species of Hyphomicrobium was searched for in the and their closest relatives was perfomed using the ae2 editor ARB database (Strunk & Ludwig, 1995), and the (Maidak et al., 1994). Phylogenetic dendrograms were position within the ' Rhodomicrobium vannielii assem- reconstructed using treeing algorithms contained in the blage' (Maidak et al., 1994) could be confirmed. PHYLIP package (Felsenstein, 1993). Bootstrap values were determined using the PHYLIP package (Felsenstein, 1993). Subsequent phylogenetic analysis was carried out using the ae2 editor. The sequences of Hypho- Nucleotide sequence accession numbers. The accession microbium strains and of Filomicrobium fusiforme were numbers of the 16s rDNAs of references strains were: aligned with each other and with representatives of Agrobacterium tumefaciens, X67223 ; Methylobacterium organophilum, M29028 ; Methy lobacterium extorquens, neighbouring taxa. A total of 1360 nucleotides were M29027 ; Phyllobacteriurn myrsinacearum, D 12789; Meso- used in the analysis, and phylogenetic trees were rhizobium loti, D12791; Rhodobium rnarinum, D30790; generated using the algorithms of De Soete (1983) and Rhodobium orientis, D30792 ; and Rhodomicrobium vannielii, those included in the PHYLIP package (Felsenstein, M34127. 1993). All trees showed very similar topologies, in that all hyphomicrobia as well as F. fusiforme formed a phylogenetically coherent group, which was most RESULTS closely related to Rhodomicrobium vannielii (around The almost complete primary structure of the 16s 90% 16s rDNA sequence similarity), while the other rDNA of eight type strains of Hyphomicrobium species reference organisms were more distantly related (be- and of Filomicrobium fusiforme as well as of four tween 87 and 89.5 YOsimilarity). The only differences in

636 International Journal of Systematic Bacteriology 48 Phylogeny of Hyphomicrobium and Filomicrobium

...... Rhodobium orientis Fig. 7- Dendrogram showing the Rhodobium marinum phylogenetic position of the genera Phyllobacterium myninacearum r-c+=FFMesorhizobium loti Hyphomicrobium and Filomicrobium among Agro bacterium turne fa ciens members of the alpha-2 subclass of the class Rhodomicrobium vannielii Proteobacteria. The tree was constructed by Hyphomicrobium facilis su bsp. ureaphilum CO-582T the neighbour-joining method (Saitou & Hyphomicrobium facilis subsp. tolerans I-551T Nei, 1987), using corrected distance values Hyphomicrobium facilis B-522 Hyphomicrobium facilis su bsp. facilis H-526T (Jukes & Cantor, 1969). The sequences Hyphomicrobium methylovorum DSM 5458T of Methylobacterium extorquens and

~ 100 I Hyphomicrobium denitrificans DSM 186gT Methylobacterium organophilum were used Filomicrobium fusiforme DSM 5304T to root the dendrogram. The position of H. Hyphom icrobium vulgare M C-7 50T zavarzinii ZV-580 corresponds fully to that Hyphomicrobium hollandicum KB-677T Hyphomicrobium aestuarii NQ-52 1grT of strain ZV-622T. Bootstrap values Hyphomicrobium zavarzinii ZV-622T (expressed as percentages of 500 replications) of 65% or more are indicated at the branch points. Bar, 5% sequence 5% divergence.

the tree topologies were observed in the branching Table 2. 16s rDNA signature nucleotides that define point of Filomicrobium fusiforme. clusters I and II of Hyphomicrobium species and their occurrence in the 165 rDNA sequence of fusiforme The type strains of the seven Hyphomicrobium species F. formed two 16s rDNA clusters in which the species ~ ~~ showed a high degree of relatedness (Fig. 1). Bootstrap Position* Cluster I Cluster I1 F.fusiforrne values of 100% indicated that these clusters are of statistical significance. Cluster I contained the type 99 A G G species of the genus H. vulgare, as well as H. 13 1-23 1 U-A C-G C-G hollandicum, H. aestuarii and H. zavarzinii. The 16s 137-226 U-A C-G U-A rDNA sequence similarities ranged between 97.8 and 138-225 A-U U-A A-U 98.8 %. Strains ZV-622T and ZV-580 had identical 16s 139-224 U-A A-U u-u rDNA sequences. Cluster I1 contained the species H. 140-223 C-G U-A U-A methylovorum, H. denitriJicans and H. facilis and its 14 1-222 G-C A-U G-C two subspecies H.facilis subsp. tolerans IFAM I-551T 144-178 A-C A-U A-U and H.facilis subsp. ureaphilum IFAM CO-582T. The 153-168 C-G C-A C-G last three strains, as well as strain IFAM B-522, shared 154-167 U-G C-G u-u 99-9YO sequence similarity. The sequence similarities LOOP187-190 UUCG GA[A/G]A GAGA of the type strains of the three species ranged from 97.3 24&286 C-G A-U C-G to 98.8 YO.Members of clusters I and I1 were separated 295-302 C-G U-A C-G by 6-4-8.0 YO sequence differences. The two clusters 241-285 U-A G-C G-C can be differentiated clearly by a set of cluster-specific 4 18-425 U-A C-G C-G nucleotides, which can be considered a molecular 422 C A A signature. The positions of these nucleotides in the 16s 50 1-544 C-G U-A C-G rDNA sequence are indicated in Table 2. 66 1-744 G-C A-U A-U 670-736 A-U G-C A-U The branching point of the F. fusiforme sequence was 818 U G G close to the bifurcation point of the two Hypho- 849 G U G microbium lineages. Although in the algorithm of De 1007-1022 u-u C-G G Soete (1983) this species grouped with members of 1008-1021 u*u C-G u-u cluster 11, it branched with members of cluster I in the 1254-1283 G-U C-G u-u neighbour-joining analysis (Saitou & Nei, 1987). The 1264-1 27 1 C-G U-A G-C finding that 16s rDNA similarity values for F. 1304 G A G fusiforme were about 2 Yo higher for members of cluster 131&1327 G-C A-U G-C I than for members of cluster I1 may be indicative of a specific relatedness to the former cluster as indicated in "E.coli position (Brosius et al., 1978). Fig. 1. This relationship is also expressed in the presence of 13 signature nucleotides that are shared between F. fusiforme and members of cluster I, while structure of the 16s rDNA of F. fusiforme, unlike those nine signatures are common to F. fusiJorme and of Hyphomicrobium species, lack a stretch between members of cluster 11. Bootstrap values of the positions 1257 and 1277. This position is also missing branching points (65 YO significance for cluster I in Rhodobium marinurn, while it is present in its closest members compared with 23 YOsignificance for cluster relative Rhodobium orientis and in the sequence of the I1 members) are too low to make a decision about the other reference organisms of the alpha-2 subclass phylogenetic neighbour of F. fusiforme. The primary included in Fig. 1.

lnternational Journal of Systematic Bacteriology 48 637 F. A. Rainey and others

DISCUSSION not be taken as an argument for either decision, as it is deep in any phylogenetic tree generated, and none of The membership of Hyphomicrobium species in the them is supported with high statistical significance. alpha-2 subclass of the class Proteobacteria confirms Also, Sittig & Hirsch (1992), while clearly separating earlier findings that were based on the analysis of 5s members of Hyphomicrobium and F.Jiliformis by the rDNA (Stackebrandt et al., 1988; Nikitin et al., 1990) results of phospholipid and fatty acids analysis, could and 16s rRNA (Stackebrandt et al., 1988; Tsuji et al., not identify specific patterns for the Hyphomicrobium 1990), determination of 16s rRNA cistron similarities species and, of all the morphological and physiological (Moore, 1977 ; Roggentin & Hirsch, 1989) and mem- properties compiled by Hirsch (1989), only the brane fatty acids (Gluckert et al., 199 1; Sittig & Hirsch, formation of rosettes or cell clumping would identify 1992), but not more than five species were included in members of cluster I, because these properties are a single study. The coherency of the genus was absent in members of cluster 11. The lack of non- supported by phage typing (Gliesche et al., 1988), molecular evidence stops us from proposing a new analysis of low-molecular-mass RNA patterns (Hofle, genus for the cluster I1 organisms. In addition, a 1990) and 16s rDNA cistron similarity studies comprehensive taxonomic revision of the genus (Roggentin & Hirsch, 1989), while 16s rRNA cata- Hyphomicrobium should await a molecular analysis of loguing showed Filomicrobium to branch within the undescribed strains for which species status has been radiation of Hyphomicrobium (Stackebrandt et al., discussed on the basis of phenotypic analysis 1988). The intragenetic structure was elucidated by the (Vedenina et al., 1990; Sittig & Hirsch, 1992), DNA latter two approaches, which clearly showed the dot-blot hybridization studies (Holm et al., 1996) and phylogenetically separate position of H. vulgare, H. morphology (Holm et al., 1996). aestuarii and H. zavarzinii on the one side and H. facilis on the other. H. hollandicum and F. fusiforme grouped intermediate to these clusters in the studies on ACKNOWLEDGEMENTS RNA cistron similarities (Roggentin & Hirsch, 1989) and 16s rRNA cataloguing (Stackebrandt et al., 1988), We wish to thank Orsola Pauker for her help in the molecular respectively. work. The distinctness of Hyphomicrobium species is demonstrated by differences of more than 1-2YO in 16s rDNA REFERENCES sequence found for the most closely related species. Boulygina, E. S., Chumakov, K. M. & Netrusov, A. 1. (1993). 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